In the state of art a plurality of scientific publications have described the construction of fusion proteins. In the majority of these cases, such fusion proteins were realised as fusions of polypeptides to the N-terminus or C-terminus of a carrier protein. Martineau et al. (Martineau, P., J. G. Guillet, et al. (1992). “Expression of heterologous peptides at two permissive sites of the MalE protein: antigenicity and immunogenicity of foreign B-cell and T-cell epitopes.” Gene 118(1): 151; Martineau, P., C. Leclerc, et al. (1996). “Modulating the immunological properties of a linear B-cell epitope by insertion into permissive sites of the MalE protein.” Mol Immunol 33(17-18): 1345-58.) have realized the insertion of a protein into permissive sites of the protein MalE and they have studied the immune response against the inserted protein.
Several authors describe the immunisation against the enterotoxins STa (heat stable enterotoxin of E. coli) via constructing N-terminal or C-terminal fusion proteins, wherein different carrier proteins are involved, in order to obtain an immune response against the STa peptide, which as such is not immunogenic. The construction of hybrid proteins by inserting the STa peptide into a permissive site of a carrier protein has not yet been described in the prior art. The β-lactamase TEM-1 also not has been used as carrier protein for the construction of hybrid or fusion proteins with STa.
The process for synthesising bifunctional proteins got underway through binding one protein to another by chemical means. Against the background of this approach, the two proteins of interest, which have different properties, are synthesised independently and then treated with a chemical so as to achieve the covalent bonding of specific chemical groups available in the proteins. The technique has helped to achieve progress in developing diagnosis tests and recombinant vaccines. However, it has several drawbacks that have prompted the scientific community to develop other options. One of the disadvantages of using chemical agents is the aspecific coupling of two target proteins, which results in a lack of uniformity in the way the two proteins are bad associated and oriented. This in turn may inactivate one of the proteins. As a result of the binding system, protein complexes are formed where the stoichiometry and composition is heterogeneous. For example, a protein X may be associated with one, two, three or more Y proteins. A binding between identical molecules (dimer-multimer) is difficult to avoid, thereby reducing the quantity of bifunctional proteins obtained. The resulting assay challenges can be intuitively understood, along with the calibration stage required to assess the sensitivity of the product with each further coupling.
Another challenge with the aspecific binding of the bridging agents is that these techniques call for large quantities of proteins in return for a reduced yield potential, thereby pushing up the costs of the finished product. A de novo synthesis of bifunctional proteins in prokaryotics or eukaryotics systems offers an alternative way of meeting these challenges. This method involves molecular biology techniques providing an opportunity to modify the structure of the coding gene for the proteins in question. However, this technology calls for a detailed knowledge of the biochemical and structural properties of the polypeptides synthesised on the basis of their manipulated sequence of nucleotides.
An initial de novo synthesis approach was adopted on the basis of fusion proteins. This involves genetically fusing coding DNA sequences for two proteins of interest to one or the other ends of the said genes. This fusion operation may apply to whole proteins, fragments of proteins or random peptides. The two proteins (or protein fragments) are then expressed in tandem by the producer organism. This technique solves the problems of difficult, insoluble, misfolding proteins. It also addresses issues related to chemical coupling (see above), even though it is not the technique by which other ones are judged in this sphere. Presenting a peptide to the ends of another protein means this protein is exposed to excessive proteolysis when the fusion protein is being produced and purified. What is more, the degrees of freedom of the fusion peptides are such that they seriously destabilise the structure of the entire fusion. The result is a total loss of biological activity.
Therefore it was object of the present invention to provide functional proteins wherein the respective carrier protein retains its activity and also the added heterologous sequence still processes its function (for example as epitope, enzyme etc.), wherein furthermore the added heterologous sequence is somehow exposed on the surface of the carrier protein which is providing the possibility that the heterologous sequence may interact with other molecules. Furthermore, it was the object of the present invention to provide the functional proteins wherein the additional heterologous sequence maintains its free dimensional structure. It was a further object that the heterologous sequence is made less susceptible to proteolysis.